The Role of Intestinal Intraepithelial Lymphocytes (IELs) in the Immunity against Enteric Viral Infections O. 1Lab
1 Eisa ,
J.
1 Chettle ,
P.
1 Tonks ,
B.
1 Blacklaws ,
of Viral Zoonotics ,University of Cambridge and
1. Introduction
M.
2 Veldhoen ,
2Babraham
J. L.
1 Heeney
Institute, Cambridge, United Kingdom
3. Results
Intraepithelial Lymphocytes (IELs) compose a large immune-cell compartment that reside within the epithelial layer of the intestine1. Although almost exclusively T-cells that originate from the bone marrow, the gut IELs compartment is extremely heterogeneous, consisting of several diverse subsets that differ in the expression of co-receptors, activation markers and T-cell receptors (TCRs) including ab-TCR and gd-TCR IELs. The combination of their unique location (above the basement membrane) with the ability to respond swiftly without the need for clonal expansion or priming qualifies them to be a potent frontline immune defence against invading pathogens. However, in spite of being described for decades, relatively little is known about how exactly they function in the intestinal mucosa especially with regard to antiviral responses.
A. Characterization of the anti-CD3 experimental model: i) Ex-vivo analysis of splenocytes, MLN and gut tissues for the expression of cytokines, and the gut sections for ISGs, showed that I.P. administration of agonistic anti-CD3 antibody results in marked systemic activation of CD3+ T-cells with the production of a number of pro-inflammatory and antiviral cytokines that culminate in an anti-viral state in the gut.
B. The role of activated IELs in the early gut immunity against MNV-O7: i) IELs are not essential for the inhibition of MNV-O7 replication mediated by anti-CD3 injection Wild-type mice
IL15R-/- mice
Anti-CD3 (I.P.) + MNV-O7 infection
Anti-CD3 protects against O7 infection compared with the control
Anti-CD3 (I.P.) + MNV-O7 infection
Wait 8 hours anti-CD3 I.P. (T cell stimulation)
Wait 40 hours
ii) Activated IELs play a minor role in the inhibition of MNVO7 viral titres mediated by anti-CD3 injection
ii) The anti-CD3-mediated inhibition of MNV-O7 viral titre is STAT1-dependent
Wild-type mice
STAT1-/- mice
Anti-CD3 (I.P.) + MNV-O7 infection
Anti-CD3 (I.P.) + MNV-O7 infection
Anti-CD3 protects against O7 infection compared with the control
TCRalpha-/- mice
Anti-CD3 (I.P.) + MNV-O7 infection
Anti-CD3 protects against O7 infection compared with the control
Anti-CD3 (I.P.) + MNV-O7 infection
???
- No TCRab T-cells (No CD4, CD8 T-cells). - Increase in the number of systemic gd T-cells. - Development of Spontaneous Inflammatory Bowel Disease.
???
???
Wait for 2 weeks to allow for reconstitution
-
Rag2-/- mice lack T and B cells. Only the injected IELs will respond to the Anti-CD3. Injected TCRγδ IEL ~ 500,000 cell per mouse (variable). Quality Control FACS of intestine: most of the mice had about 50% of the normal number of TCRγδ IEL.
We were able to show that in vivo activation of CD3+ T-cells in wild-type mice, by intraperitoneal injection of agonistic anti-CD3 antibody, resulted in induction of a strong, STAT1-dependent, antiviral state in the gut mucosa that significantly reduced the establishment of MNV-O7 infection. Clearly, this protective effect cannot be attributed to the activation of the T-cells in the gut only, as the anti-CD3 antibody has a marked systemic, ‘cytokine storm’-like effect as indicated by the activation of lymphocytes in the spleen and MLNs. In order to minimize this systemic effect, future work will include assessment of oral administration of the anti-CD3 antibodies. Analysis of the Il15r-/-, Tcra-/- and the preliminary Rag2-/- adoptive transfer experiments suggests that, at least in the context of our experimental conditions, the activated IELs are not essential and play a minor role in the anti-CD3-mediated protection against MNV-O7. Future work will include attempts to improve the Rag2-/- adoptive-transfer experimental model by, for example, addition of B cell prior to MNV-O7 infection.
5. References 1. Cheroutre, H., Lambolez, F. & Mucida, D. The light and dark sides of intestinal intraepithelial lymphocytes. Nat.Rev.Immunol. 11, 445–56 (2011). 2. Li, Y. et al. Exogenous stimuli maintain intraepithelial lymphocytes via aryl hydrocarbon receptor activation. Cell 147, 629–40 (2011). 3. Swamy, M. et al. Intestinal intraepithelial lymphocyte activation promotes innate antiviral resistance. Nat. Commun. 6, 7090 (2015). 4. Karst, S. M., et al. STAT1-dependent innate immunity to a Norwalk-like virus. Science 299, 1575–8 (2003).
Analysis of gut sections for MNV titres
Oral infection MNV-O7
- Virus titres were estimated by qRT-PCR or by measuring live infectious virus
titres using a TCID50 assay. - Anti-CD3 effects: wild-type mice were injected (I.P.) with 25µg of either anti-
CD3 (+) or isotype control (-) antibodies. 8 hours later the mice were sacrificed and spleen, mesenteric LN and gut sections (duodenum and ileum) were collected and analysed by flow cytometry (for splenocytes) and for gene expression using qRT-PCR (MLN and gut sections).
AntiCD3 + MNV-O7
4. Discussion
Wild-type mice
The role of IELs was assessed by comparing the antiviral immune response to a standardized dose of MNV-O7. An infectious viral dose of 5 x 106 TCID50 was administered orally to wild-type and/or specific knockout mouse strains, 8-hours after intra-peritoneal injection of either agonistic anti-CD3 (25µg) or isotype control antibodies. Mice were sacrificed 40-hours after virus challenge to determine the degree of protection observed at different sites in the small intestine
Adoptive-Transfer of FACS-sorted IEL
???
Recent data indicate the capability of activated IELs to play a powerful role in early immunity against enteric viral infections by production of a large amount of type I and type-III Interferons upon activation3.
2. Experimental Model and Methods
Rag2-/- mice
- Deficiency of all subtypes of IEL. - Completely lack NK cells. - Reduction in NKT cells and memory-phenotype CD8+ cells.
Splenocytes
Using a mouse-model of enteric viral (Murine Norovirus, strain O7, MNV-O7) infection the main aim of this project is to analyse and dissect the role of the different IEL subsets in early innate immunity against gastrointestinal viral infection.
iii) Adoptive Transfer of Gamma/Delta TCR+ IELs into Rag2-/- mice.
6. Acknowledgements * ~ P value < 0.05 ** ~ P value < 0.01 n.s. ~ non-significant
Special thanks to the Islamic Development Bank, the Cambridge International Trust, St. Edmunds College and BBSRC for funding this project.